Isoxazoline Compounds Having MIF Antagonist Activity

ABSTRACT

Methods of use and pharmaceutical compositions for a genus of low molecular weight compounds comprising optionally substituted isoxazoline ring systems that act as inhibitors of MIF (macrophage migration inhibitory factor) are disclosed. Specifically, the compounds are useful for treating a variety of diseases involving inflammatory activity or pro-inflammatory cytokine responses, such as autoimmune diseases (including rheumatoid arthritis, insulin-dependent diabetes, multiple sclerosis, graft versus host disease, lupus syndromes), asthma, arthritis, ARDS, psoriasis, interleukin-2 toxicity, proliferative vascular disease, and various forms of sepsis and septic shock, and other conditions characterized by underlying MIF responses including, for instance, tumor growth and neovascularization (angiogenesis).

This application claims priority from U.S. Provisional Application Ser.No. 60/296,478 filed Jun. 8, 2001. The entirety of that provisionalapplication is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides a genus of optionally substitutedisoxazoline compounds and related methods of use and pharmaceuticalcompositions. The compounds have MIF (macrophage migration inhibitoryfactor) antagonist activity. Specifically, the MIF antagonists areuseful in methods for treating a variety of diseases involvinginflammatory activity or pro-inflammatory cytokine responses, such asautoimmune diseases, asthma, arthritis, multiple sclerosis, ARDS (acuterespiratory distress syndrome) and various forms of sepsis and septicshock, and other conditions characterized by underlying MIF responsesincluding, for instance, tumor growth and neovascularization.

2. Background of the Technology

Macrophage migration inhibitory factor (MIF) is one of the earliestdescribed cytokines, and is an immunoregulatory protein with a widevariety of cellular and biological activities (for reviews see: Swope,et al., Rev. Physiol. Biochem. Pharmacol. 139, 1-32 (1999); Metz, etal., Adv. Immunol. 66, 197-223 (1997); and Bucala, FASEB J. 14,1607-1613 (1996)). Originally, MIF was found to be secreted by activatedlymphoid cells, to inhibit the random migration of macrophages, and tobe associated with delayed-type hypersensitivity reactions (George etal., Proc. Soc. Exp. Biol. Med., 111, 514-521 (1962); Weiser, et al., J.Immunol. 126, 1958-1962 (1981); Bloom et al., Science, 153:80-82 (1966);David, Proc. Natl. Acad. Sci. USA, 56, 72-77 (1966). MIF was also shownto enhance macrophage adherence, phagocytosis and tumoricidal activity(Nathan et al., J. Exp. Med., 137, 275-288 (1973); Nathan, et al., J.Exp. Med., 133, 1356-1376 (1971); Churchill, et al., J. Immunol., 115,781-785 (1975)). Unfortunately, many of the early MIF studies usedmixed-culture supernatants that were shown later to contain othercytokines, such as IFN-γ and IL-4, that also have macrophage migrationinhibitory activity (McInnes, et al., J. Exp. Med., 167, 598-611 (1988);Thurman, et al., J. Immunol., 134, 305-309 (1985)). The availability ofrecombinant MIF has allowed for confirmation of these biologicalactivities, and for the identification of additional activities.

Recombinant human MIF was originally cloned from a human T cell library(Weiser, et al., Proc. Natl. Acad. Sci. USA, 86, 7522-7526 (1989)), andwas shown to activate blood-derived macrophages to kill intracellularparasites and tumor cells in vitro, to stimulate IL-1β and TNFαexpression, and to induce nitric oxide synthesis (Weiser, et al., J.Immunol., 147, 2006-2011 (1991); Pozzi, et al., Cellular Immunol., 145,372-379 (1992); Weiser, et al., Proc. Natl. Acad. Sci. USA, 89,8049-8052 (1992); Cunha, et al., J. Immunol., 150, 1908-1912 (1993)).While the conclusions available from several of these early reports areconfounded by the presence of a bioactive mitogenic contaminant in therecombinant MIF preparations used, the potent pro-inflammatoryactivities of MIF have been established in other studies that do notsuffer from this complicating factor (reviewed in Bucala, The FASEB,Journal 10, 1607-1613 (1996)).

More recent MIF studies have capitalized on the production ofrecombinant MIF in purified form as well as the development ofMIF-specific polyclonal and monoclonal antibodies to establish thebiological role of MIF in a variety of normal homeostatic andpathophysiological settings (reviewed in Rice, et al., Annual Reports inMedicinal Chemistry, 33, 243-252 (1998)). Among the most importantinsights of these later reports has been the recognition that MIF notonly is a cytokine product of the immune system, but also is ahormone-like product of the endocrine system, particularly the pituitarygland. This work has underscored the potent activity of MIF as acounter-regulator of the anti-inflammatory effects of theglucocorticoids (both those endogenously released and thosetherapeutically administered), with the effect that the normalactivities of glucocorticoids to limit and suppress the severity ofinflammatory responses are inhibited by MIF. The endogenous MIF responseis thus seen as a cause or an exacerbative factor in a variety ofinflammatory diseases and conditions (reviewed in Donnelly, et al.,Molecular Medicine Today, 3, 502-507 (1997)).

MIF is now known to have several biological functions beyond itswell-known association with delayed-type hypersensitivity reactions. Forexample, as mentioned above, MIF released by macrophages and T cellsacts as a pituitary mediator in response to physiological concentrationsof glucocorticoids (Bucala, FASEB J., 14, 1607-1613 (1996)). This leadsto an overriding effect of glucocorticoid immunosuppressive activitythrough alterations in TNF-α, IL-1B, IL-6, and IL-8 levels. Additionalbiological activities of MIF include the regulation of stimulated Tcells (Bacher et al., Proc. Natl. Acad. Sci. USA, 93, 7849-7854 (1996)),the control of IgE synthesis (Mikayama, et al., Proc. Natl. Acad. Sci.USA, 90, 10056-10060 (1993)), the functional inactivation of the p53tumor suppressor protein (Hudson, et al., J. Exp. Med., 190, 1375-1382(1999)), the regulation of glucose and carbohydrate metabolism (Sakaue,et al., Mol. Med., 5, 361-371 (1999)), and the attenuation of tumor cellgrowth and tumor angiogenesis (Chesney, et al., Mol. Med., 5, 181-191(1999); Shimizu, et al., Biochem. Biophys. Res. Commun., 264, 751-758(1999)).

MIF shares significant sequence homology (36% identity) withD-dopachrome tautomerase. This led to the discovery that MIF hasenzymatic activity and catalyzes the tautomerization of thenon-physiological substrates D-dopachrome (Rosengren, et al., Mol. Med.,2, 143-149 (1996)) and L-dopachrome methyl ester (Bendrat, et al.,Biochemistry, 36, 15356-15362 (1997). Additionally, phenylpyruvic acidand p-hydroxyphenylpyruvic acid (Rosengren, et al., FEBS Letter, 417,85-88 (1997)), and 3,4-dihydroxyphenylaminechrome andnorepinephrinechrome (Matsunaga, et al., J. Biol. Chem., 274, 3268-3271(1999), are MIF substrates, although it is not known if tautomerizationof any of these agents comprises a natural function for MIF.

The three-dimensional crystal structure of human MIF reveals that theprotein exists as a homotrimer (Lolis, et al., Proc. Ass. Am. Phys.,108, 415-419 (1996) and is structurally related to 4-oxalocrotonatetautomerase, 5-carboxymethyl-2-hydroxymuconate, chorismate mutase, andto D-dopachrome tautomerase (Swope, et al., EMBO J., 17, 3534-3541(1998); Sugimoto, et al., Biochemistry, 38, 3268-3279 (1999). Recently,the crystal structure has been reported for the complex formed betweenhuman MIF and p-hydroxyphenylpyruvic acid (Lubetsky, et al.,Biochemistry, 38, 7346-7354 (1999). It was found that the substratebinds to a hydrophobic cavity at the amino terminus and interacts withPro-1, Lys-32, and Ile-64 in one of the subunits, and with Tyr-95 andAsn-97 in an adjacent subunit. Similar interactions between murine MIFand (E)-2-fluoro-p-hydroxycinnamate have been reported (Taylor, et al.,Biochemistry, 38, 7444-7452 (1999)). Solution studies using NMR providefurther evidence of the interaction between p-hydroxyphenylpyruvic acidand Pro-1 in the amino-terminal hydrophobic cavity (Swope, et al., EMBOJ., 17, 3534-3541 (1998)).

Mutation studies provide convincing evidence that Pro-1 is involved inthe catalytic function of MIF. Deletion of Pro-1 or replacement of Pro-1with Ser (Bendrat et al., Biochemistry, 36, 15356-15362 (1997)), Gly(Swope, et al., EMBO J., 17, 3534-3541 (1998)), or Phe(Hermanowski-Vosatka, et al., Biochemistry, 38, 12841-12849 (1999)), andaddition of an N-terminal peptide tag to Pro-1 (Bendrat, et al.,Biochemistry, 36, 15356-15362 (1997)) abrogated the catalytic activityof MIF in assays using L-dopachrome methyl ester andp-hydroxyphenylpyruvic acid. A similar loss in activity was found byinserting Ala between Pro-1 and Met-2 (Lubetsky et al., Biochemistry,38, 7346-7354 (1999). The connection between the enzymatic andbiological activities, however, remains unclear. The Pro to Ser MIFmutant showed glucocorticoid counter-regulatory activity (Bendrat, etal., Biochemistry, 36, 15356-15362 (1997)) and was fully capable, as wasthe Pro to Phe mutant, of inhibiting monocyte chemotaxis(Hermanowski-Vosatka et al., Biochemistry, 38, 12841-12849 (1999). Incontrast, the Pro to Gly MIF mutant was greatly impaired in its abilityto stimulate superoxide generation in activated neutrophils (Swope etal., EMBO J., 17, 3534-3541 (1998). These results suggest that thebiological activity of enzymatically inactive MIF mutants may bedependent not only on the nature of the mutation, but also on the assaythat is used to assess biological function.

There is a need in the art to discover and develop small organicmolecules that function as MIF inhibitors (e.g., antagonists) andfurther posses the benefits of small organic molecule therapeuticsversus larger, polymeric protein (e.g., antibody) and nucleic acid-based(e.g., anti-sense) therapeutic agents. The therapeutic potential of lowmolecular weight MIF inhibitors is substantial, given the activities ofanti-MIF antibodies in models of endotoxin- and exotoxin-induced toxicshock (Bernhagen et al., Nature, 365, 756-759 (1993); Kobayashi et al.,Hepatology, 29, 1752-1759 (1999); Calandra et al., Proc. Natl. Acad.Sci. USA., 95, 11383-11388 (1998); and Makita et al., Am. J. Respir.Crit. Care Med. 158, 573-579 (1998), T-cell activation (Bacher et al.,Proc. Natl. Acad. Sci. USA., 93, 7849-7854 (1996), autoimmune diseases(e.g., graft versus host disease, insulin-dependent diabetes, andvarious forms of lupus) including rheumatoid arthritis (Kitaichi, etal., Curr. Eye Res., 20, 109-114 (2000); Leech, et al., ArthritisRheum., 42, 1601-1608 (1999), wound healing (Abe, et al., Biochim.Biophys. Acta, 1500, 1-9 (2000), and angiogenesis (Shimizum, et al.,Biochem. Biophys. Res. Commun., 264, 751-758 (1999). Low molecularweight anti-MIF drugs exhibiting such activities may offer clinicaladvantages over neutralizing antibodies and nucleic acid-based agentsbecause they may be orally active or generally more easily administered,have better bioavailabilities, have improved biodistributions, andshould be much cheaper to produce. Prior to the present invention, theonly published report of potent low molecular weight MIF inhibitorsconcerned some commonly found long chain fatty acids that reversiblyinhibited the dopachrome tautomerase activity of mouse MIF (Bendrat etal., Biochemistry, 36, 15356-15362 (1997). These fatty acids were nevertested for their effects in biological assays of MIF activity.

U.S. Pat. No. 4,933,464 to Markofsky discloses a process for forming3-phenylisoxazolines and 3-phenylisoxazoles and related products.

U.S. Pat. No. 6,114,367 to Cohan et al. discloses isoxazoline compoundswhich are inhibitors of tumor necrosis factor (TNF). The isoxazolinecompounds are said to be useful for inhibiting TNF in a mammal in needthereof and in the treatment or alleviation of inflammatory conditionsor disease. Also disclosed are pharmaceutical compositions comprisingsuch compounds.

Curuzu et al., Collect. Czech. Chem. Commun., 56: 2494-2499 (1991)discloses 3-substituted phenyl-4,5-dihydroisoxazoleneacetic acids,including 3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic acid and3-(4-methoxyphenyl)-4,5-dihydro-5-isoxazolineacetic acid, and shows thatthe first of these two compounds is devoid of antiinflammatory activity,while the second is dramatically reduced in such activity compared tothe parent compound that was unsubstituted in the para position of thephenyl ring, in a carageenin-induced edema assay in the rat paw.

Wityak et al., J. Med. Chem., 40: 50-60 (1997) discloses isoxazolineantagonists of the glycoprotein IIb/IIIa receptor.

Eichenger, et al., Synth. Commun. 27 (16): 2733-2742 (1997) discloses[3-(4-methoxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-acetic acid.

Eichinger, et al., Synth. Commun. 28(13): 2457-2466 (1998) discloses[3-(4-methoxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-acetic acid and themethyl ester thereof.

Kleinman, et al., “Striking effect of hydroxamic acid substitution onthe phosphodiesterase type 4 (PDE4) and TNF alpha inhibitory activity oftwo series of rolipram analogues: implications for a new active sitemodel of PDE4”. J. Med. Chem. 41(3): 266-270 (1998), discloses interalia the following compounds:[3-(3-cyclopentyloxy-4-methoxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-aceticacid and the methyl ester thereof, as well as[3-(3-cyclopentyloxy-4-methoxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-N-hydroxy-acetamide.

U.S. application Ser. No. 09/625,829, filed Jul. 26, 2000, which ishereby incorporated herein by reference in its entirety, disclosesquinone-related compounds having MIF inhibitor activity. U.S.application Ser. No. 09/699,258, filed Oct. 27, 2000, which is herebyincorporated herein by reference in its entirety, discloses aminoacid/benzaldehyde Schiff base compounds having MIF inhibitor activity.

SUMMARY OF THE INVENTION

The enzyme activity (tautomerase) of MIF and the substrates it acceptsprovide an enzymatic activity assay for designing low molecular weightagents that bind to MIF and disrupt its biological activity. The presentinvention provides methods of use for a genus of such compounds havingisoxazoline structures.

The present invention provides a method for treating inflammatorydisorders including, but not limited to, arthritis, proliferativevascular disease, ARDS (acute respiratory distress syndrome),cytokine-mediated toxicity, sepsis, septic shock, psoriasis,interleukin-2 toxicity, asthma, MIF-mediated conditions, autoimmunedisorders (including but not limited to, rheumatoid arthritis,insulin-dependent diabetes, multiple sclerosis, graft versus hostdisease, lupus syndromes), tumor growth or angiogenesis, or anycondition characterized by local or systemic MIF release or synthesis,comprising administering an effective amount of a compound of Formula I,wherein Formula I is:

wherein:

R₁₋₄ are, independently, R, halo, N₃, CN, OH, NRR′, or SH;

R and R′ are, independently, H or C₁₋₆ alkyl;

X is R, halo, N₃, CN, OR, NRR′, SH, ═O, ═CH₂, or A;

A is a substituted or unsubstituted aromatic ring;

Y is R, NRR′, NRR″ or (CH₂)_(n)-A;

Z is R, OR, OR″, NRR′, NRR″, or A;

R″ is a saturated or unsaturated, straight or branched chain C₂-C₁₈, andn is 0 or 1.

Preferably, the compound is a p-hydroxyphenyl-isoxazoline-containingcompound, wherein each of R, R¹⁻⁴, X and Y is H or —CH₂-A, and Z is OR.More preferably, the compound is an ester of(R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic, particularlythe acid methyl ester thereof (identified as “ISO-1” herein) which isalso known as, p-hydroxyphenol-isoxazoline methyl ester. Still morepreferably the compound is an ester of2-[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-3-phenyl-propinoicacid, particularly the methyl ester thereof (identified as “ISO-2”).

The present invention further provides a pharmaceutical compositioncomprising a compound having an isoxazoline moiety, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier or diluant, wherein the composition comprises aneffective amount of a compound of Formula I, wherein Formula I is:

wherein:

R₁₋₄ are, independently, R, halo, N₃, CN, OH, NRR′, or SH;

R and R′ are, independently, H or C₁₋₆ alkyl;

X is R, halo, N₃, CN, OR, NRR′, SH, ═O, ═CH₂, or A;

A is a substituted or unsubstituted aromatic ring;

Y is R, NRR′, NRR″ or (CH₂)_(n)-A;

Z is R, OR, OR″, NRR′, NRR″, or A;

R″ is a saturated or unsaturated, straight or branched chain C₂-C₁₈;

and n is 0 or 1.

with any or all of the following provisos:(i) when Z=OCH₃, at least one of R, R₁₋₄, X and Y is not H;(ii) when R₁=R₂=tert-butyl and each is ortho to OR, at least one of R,R₃, R₄, X and Y is not H or Z is not OCH₃;(iii) when Z=OH and R=H or methyl, at least one of R₄, X and Y is not H(i.e., the compound is not[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-acetic acid or[3-(4-methoxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-acetic acid); and(iv) when Z=OCH₃, and R=methyl, at least one of R₁₋₄, X and Y is not H(i.e., the compound is not[3-(4-methoxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-acetic acid methylester).

The present invention also provides a pharmaceutical compositioncomprising a compound having an isoxazoline moiety, and apharmaceutically acceptable carrier, wherein the compound forms a stableinteraction with at least one amino acid residue of an MIF protein.Preferably, the interaction occurs at or near the active site of thetautomease activity of the MIF protein. Preferably, the compound is ahydroxyphenyl-isoxazoline-containing compound. More preferably, thecompound is an ester of(R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic acid,particularly the methyl ester (ISO-1). Still more preferably thecompound is and ester of2-[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-3-phenyl-propinoicacid, particularly the methyl ester (ISO-2).

The present invention provides a method for treating inflammatorydisorders (including, but not limited to, arthritis, proliferativevascular disease, ARDS (acute respiratory distress syndrome),cytokine-mediated toxicity, sepsis, septic shock, psoriasis,interleukin-2 toxicity, asthma, MIF-mediated conditions, autoimmunedisorders (including but not limited to, rheumatoid arthritis,insulin-dependent diabetes, multiple sclerosis, graft versus hostdisease, lupus syndromes), tumor growth or angiogenesis, or anycondition characterized by local or systemic MIF release or synthesis,comprising administering an effective amount of a compound having aisoxazoline moiety, wherein the compound forms a stable interaction withMIF protein. Preferably, the compound is a isoxazoline-containingcompound or an hydroxyphenyl-isoxazoline-containing compound. Morepreferably, the compound is an ester of(R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic acid,particularly the methyl ester (ISO-1). Still more preferably thecompound is and ester of2-[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-3-phenyl-propinoicacid, particularly the methyl ester (ISO-2).

In accordance with the activity of MIF to interfere with theanti-inflammatory effects of steroids (such as the anti-inflammatoryglucocorticoids), the compounds of Formula I find further utility toenhance the activity and therapeutic benefits of both endogenouslyarising and exogenously administered steroidal anti-inflammatory agents.Such benefits may, in some cases, be most evident by a reduced need forsteroid therapy (e.g., lower dose amount or frequency; less potentagent; reduced need for systemic administration) or by reducedside-effects associated with steroid administration. The benefits ofadministering an MIF inhibitor (and specifically a compound of FormulaI) may be realized as a monotherapy, using only the MIF inhibitor of thepresent invention, or as a combination therapy with additionalanti-inflammatory agents, including especially, but without limitation,an anti-inflammatory steroid. Such combination therapy may be achievedthrough administration of a single formulation or pharmaceuticalcomposition that combines the MIF inhibitor (particularly an inhibitorof Formula I) with at least one other anti-inflammatory agent (which maybe a steroidal or a non-steroidal anti-inflammatory agent), or throughadministration of separate formulations or pharmaceutical compositionsin conjunction with each other, or both.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the synthesis of p-hydroxyphenyl-isoxazoline methyl ester.

FIG. 2 shows modifications of isoxazoline 4 (ISO-1), includingoxidation, reduction and methylation.

FIG. 3 shows that compound 4 (ISO-1) (FIG. 1) inhibits MIFglucocorticoid regulating activity. The capacity of MIF proteins toregulate glucocorticoid suppression of TNF production in monocytes inthe presence of different concentrations of compound 4 was assayed asdescribed previously (Bendrat, et al., Biochemistry, 36, 15356-15362(1997). The monocytes were purified from peripheral blood by adherenceand 1×10⁶ cells/well pre-incubated for 1 hr with dexamethasone (10⁻⁸ M),MIF (100 ng/ml native MIF), and/or compound 4 in various concentrationsshown in the figure, before the addition of 0.5 μg/ml LPS (E. coli0111:B4, Sigma Chemical Co.). For the cultures corresponding to thethird bar from the left, an amount of solvent (DMSO) equal to that usedfor solubilization of compound 4 at 20 μM (fifth and sixth bars fromleft) was added. Cell culture supernatants were collected after 16 hrsand secreted TNFα quantified by a standard, commercially availableELISA. Compound 4 did not affect cell viability, as assessed by MTTreduction by standard methods known in the art. Data shown are mean±SDof triplicate wells in a representative experiment that was repeatedtwice, with similar results.

FIG. 4 shows a retrosynthetic approach to2-[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-3-phenyl-propinoicacid methyl ester (ISO-2).

FIG. 5 shows synthesis of functionalized isoxazoline derivatives.

DESCRIPTION OF THE INVENTION

Based on structural similarities to amino acid Schiff base-typecompounds described previously (see U.S. application Ser. No.09/699,258, supra), the present inventors explored additionalphenylimine scaffolds as pharmacophores for potential MIF antagonists.Several representative phenylimine compounds were synthesized and testedas inhibitors of the dopachrome tautomerase activity of MIF, and it wasconcluded that the isoxazolines represented an attractive scaffold forfurther attention. Thus,(S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole-acetic acid methylester presents certain structural elements that appeared to be importantfor binding to the MIF tautomerase active site, based on previousanalysis by the inventors of phenylpyruvate:MIF co-crystallization andSAR results with the amino acid/benzaldehyde Schiff base type compoundsU.S. application Ser. No. 09/699,258 (e.g., p-hydroxylated phenylscaffold bearing an imine bond with an associated distal esterfunction).

Accordingly, the present invention provides a new class of potential MIFinhibitors related to isoxazoline which, unlike quinone-relatedinhibitors previously disclosed (U.S. application Ser. No. 09/699,258),may be suitable to neutralize both endogenous and exogenous MIF. Inparticular, isoxazoline was found as a racemic mixture to inhibit bothMIF tautomerase and immunoregulatory activities with an IC₅₀ of 5.0micromolar. Analysis of the co-crystal of MIF and isoxazoline 4 revealedthe binding of only the S-enantiomer.

The present invention therefore provides a genus of MIF inhibitorcompounds. Compounds in this genus are identified as MIF inhibitorsbecause they inhibit MIF enzymatic activity in vitro. MIF catalyzes thetautomerization of a dopachrome-related MIF substrate to a colorlessproduct. Unless specifically indicated to the contrary, references madeherein to an inhibitory concentration (e.g., IC₅₀ or other activityindex) refer to the inhibitory activity of a test compound in an MIFtautomerase assay (as further described in detail below, and in Bendrat,et al., Biochemistry, 36, 15356-15362 (1997).

MIF Tautomerase Activity

MIF catalyzes a tautomerization (i.e., keto-enol isomerization) reaction(Rosengren, et al., Molecular Medicine, 2, 143-149 (1996). The mostactive substrate identified is a non-physiological D-isomer ofdopachrome. This reaction predicts therapeutic MIF inhibitors (seepending U.S. application Ser. No. 08/602,929, filed Feb. 16, 1996, andU.S. application Ser. No. 09/699,258, filed Oct. 27, 2000, thedisclosures of which are incorporated herein by reference in theirentirety). Inhibition of MIF tautomerase activity is predictive ofinhibition of MIF biological activity.

A method for performing an assay for MIF dopachrome tautomerase activitybegins with the preparation and oxidation of a DOPA-related substrateprecursor, such as L-3,4-dihydroxyphenylalanine methyl ester. Thisoxidation with sodium periodate generates the corresponding dopachromederivative (e.g., L-3,5-dihydro-6-hydroxy-5-oxo-2H-indole-2-carboxylicacid methyl ester (“dopachrome methyl ester”) that is orange-colored andcomprises a convenient substrate for use in a photometric assay for theenzymatic activity of MIF as a tautomerase. MIF (typically a purifiedpreparation of recombinant MIF at a final concentration of 50-1000ng/ml) addition causes the rapid tautomerization of the coloreddopachrome substrate to a colorless 5,6-dihydroxyindole-2-carboxylicacid methyl ester product. The enzymatic activity of MIF is measured asthe rate of de-colorization of the colored solution of thedopachrome-related substrate in a suitable buffer, typically at a time20 seconds after addition of the final assay component and mixing. Theabsorbance is measured at about 475 nm (or 550 nm for substrateconcentrations in excess 0.5 nM). A test compound may be included in theassay solution such that the effect of the test compound on MIFtautomerase activity (i.e., as an inhibitor) may be measured by notingthe change in kinetics of substrate tautomerization compared to controlassays performed in the absence of the test inhibitor compound. Inparticular, the MIF tautomerase assay may be conducted essentially asfollows:

L-3,4-dihydroxyphenylalanine methyl ester (e.g., Sigma D-1507) is adopachrome substrate precursor, and is prepared as a 4 mM solution in ddH₂O. Sodium periodate is prepared as an 8 mM solution in dd H₂O. AssayBuffer (50 mM potassium phosphate/1 mM EDTA, pH 6.0) is prepared.Purified recombinant MIF is prepared in 150 mM NaCl/20 mM Tris buffer(pH 7.4) as a stock solution convenient to supply MIF at a finalconcentration of about 700 ng/ml. Immediately prior to initiating theassay, 3.6 ml dopachrome substrate precursor solution, 2.4 ml periodatesolution and 4.0 ml Assay Buffer are combined into a homogeneous mixture(this preparation of dopachrome substrate is suitable for assay useafter 1 min and for about 30 min thereafter). Test compound (typicallyprepared as a concentrated stock in DMSO) and MIF are then combined with0.7 ml Assay Buffer plus 0.3 ml dopachrome substrate solution to providethe desired final concentration of the test compound in a homogeneousmixture, and the optical density (absorbance) of this assay mixture ismonitored at 475 nm. Typically, OD₄₇₅ is recorded every 5 sec for 0-60sec, and the OD₄₇₅ for a given time point is compared to parallel assayswhere MIF is not added or the test compound is omitted. Inhibition ofMIF tautomerase activity by the test compounds is determined byinhibition of the de-colorization of the assay mixture, often at the 20sec time point. IC₅₀ values for compounds with MIF tautomeraseinhibitory activity, corresponding to the concentration of inhibitorthat would inhibit MIF tautomerase activity by 50%, are determined byinterpolation of the results from MIF tautomerase assays at severaldifferent inhibitor concentrations. These IC₅₀ values provide areasonable correlation between MIF enzymatic inhibitory activity of thetest compounds, and inhibition of the biological activity of MIF (seebelow).

Methods of Treatment and Pharmaceutical Compositions

The MIF tautomerase assay was used in Example 1 shows that certainisoxazoline-containing compounds inhibit MIF enzymatic activity. Example2 shows that certain isoxazoline-based compounds not only specificallyinhibit MIF enzymatic activity (i.e, tautomerase), but also inhibit MIFimmunoregulatory activities as measured in assays of MIF biologicalactivity. Finally, Example 3 shows the co-crystallization structure ofthe MIF:isoxazoline complex, showing that three molecules of theS-isomer only of p-hydroxyphenyl-isoxazoline bind stoichiometrically tothe three active sites within the MIF trimer. These X-ray data representvaluable information for predicting the next generation of MIF activityinhibitors.

These data provide a reasonable correlation between the MIF tautomeraseenzymatic assay and MIF antagonism in a biological assay. Collectively,these data show that inhibition by a compound in the MIF tautomeraseassay is predictive of its potential therapeutic use in inhibiting MIFbiological activity.

Accordingly, the present invention provides a method for inactivatingenzymatic and biological activity of human MIF comprising contacting thehuman MIF with a compound, or combination of compounds, having anisoxazoline moiety that forms a stable interaction with at least oneamino acid residue of the human MIF. Preferably, the interaction occursat or near the active site of the tautomease activity of the MIFprotein. Preferably, the stable interaction is between the isoxazolinemoiety of the isoxazoline-containing compound and the N-terminal prolineresidue of the human MIF. Preferably the compound is aisoxazoline-containing compound of Formula I, wherein Formula I is:

wherein:

R₁₋₄ are, independently, R, halo, N₃, CN, OH, NRR′, or SH;

R and R′ are, independently, H or C₁₋₆ alkyl;

X is R, halo, N₃, CN, OR, NRR′, SH, ═O, ═CH₂, or A;

A is a substituted or unsubstituted aromatic ring (substituted, forinstance, with one or more groups selected from R, halo, N₃, CN, OH,NRR′, or SH);

Y is R, NRR′, NRR″ or (CH₂)_(n)-A;

Z is R, OR, OR″, NRR′, NRR″, or A;

R″ is a saturated or unsaturated, straight or branched chain C₂-C₁₈;

and n is 0 or 1.

Preferably, the compound is a p-hydroxyphenyl-isoxazoline-containingcompound, wherein each of R, R₁₋₄, X, and Y is H or —CH₂-A, and Z is OR.More preferably, the compound is an ester of(R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic acid,particularly the methyl ester (ISO-1). Still more preferably thecompound is and ester of2-[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-3-phenyl-propinoicacid, particularly the methyl ester (ISO-2).

The present invention also provides a method for treating inflammatorydisorders including, but not limited to, arthritis, proliferativevascular disease, ARDS (acute respiratory distress syndrome),cytokine-mediated toxicity, sepsis, septic shock, psoriasis,interleukin-2 toxicity, asthma, MIF-mediated conditions, autoimmunedisorders (including, but not limited to, rheumatoid arthritis,insulin-dependent diabetes, multiple sclerosis, graft versus hostdisease, lupus syndromes), tumor growth or angiogenesis, or anycondition characterized by local or systemic MIF release or synthesis,comprising administering an effective amount of a compound of Formula I,wherein Formula I is:

wherein:

R₁₋₄ are, independently, R, halo, N₃, CN, OH, NRR′, or SH;

R and R′ are, independently, H or C₁₋₆ alkyl;

X is R, halo, N₃, CN, OR, NRR′, SH, ═O, ═CH₂, or A;

A is a substituted or unsubstituted aromatic ring;

Y is R, NRR′, NRR″ or (CH₂)_(n)-A;

Z is R, OR, OR″, NRR′, NRR″, or A;

R″ is a saturated or unsaturated, straight or branched chain C₂-C₁₈; Pand n is 0 or 1.

Preferably, the compound is a p-hydroxyphenyl-isoxazoline-containingcompound, wherein each of R, R¹⁻⁴, X and Y is H or —CH₂-A, and Z is OR.More preferably, the compound is an ester of(R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic acid,particularly the methyl ester (ISO-1). Still more preferably thecompound is and ester of2-[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-3-phenyl-propinoicacid, particularly the methyl ester (ISO-2).

The present invention further provides a pharmaceutical compositioncomprising a compound having an isoxazoline moiety, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier or diluant, wherein the composition comprises aneffective amount of a compound of Formula I, wherein Formula I is:

wherein:

R₁₋₄ are, independently, R, halo, N₃, CN, OH, NRR′, or SH;

R and R′ are, independently, H or C₁₋₆ alkyl;

X is R, halo, N₃, CN, OR, NRR′, SH, ═O, ═CH₂, or A;

A is a substituted or unsubstituted aromatic ring;

Y is R, NRR′, NRR″ or (CH₂)_(n)-A;

Z is R, OR, OR″, NRR′, NRR″, or A;

R″ is a saturated or unsaturated, straight or branched chain C₂-C₁₈;

and n is 0 or 1.

with any or all of the following provisos:(i) when Z=OCH₃, at least one of R, R₁₋₄, X and Y is not H;(ii) when R₁=R₂=tert-butyl and each is ortho to OR, at least one of R,R₃, R₄, X and Y is not H or Z is not OCH₃;(iii) when Z=OH and R=H or methyl, at least one of R₁₋₄, X and Y is notH (i.e., the compound is not[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-acetic acid or[3-(4-methoxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-acetic acid); and(iv) when Z=OCH₃, and R=methyl, at least one of R₁₋₄, X and Y is not H(i.e., the compound is not[3-(4-methoxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-acetic acid methylester).

The present invention further provides a method for treatinginflammatory disorders including, but not limited to, arthritis,proliferative vascular disease, ARDS (acute respiratory distresssyndrome), cytokine-mediated toxicity, sepsis, septic shock, psoriasis,interleukin-2 toxicity, asthma, MIF-mediated conditions, autoimmunedisorders (including, but not limited to, rheumatoid arthritis,insulin-dependent diabetes, multiple sclerosis, graft versus hostdisease, lupus syndromes), tumor growth or angiogenesis, or anycondition characterized by local or systemic MIF release or synthesis,comprising administering an effective amount of a compound having anisoxazoline moiety, wherein the isoxazoline moiety forms a stablecovalent interaction with at least one amino acid residue of an MIFprotein. Preferably, the interaction occurs at or near the active siteof the tautomease activity of the MIF protein. Preferably, the compoundis a isoxazoline-containing compound or anhydroxyphenyl-isoxazoline-containing compound. More preferably, thecompound is an ester of(R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic acid,particularly the methyl ester (ISO-1). Still more preferably thecompound is and ester of2-[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-3-phenyl-propinoicacid, particularly the methyl ester (ISO-2).

The present invention also provides a pharmaceutical compositioncomprising a compound having a isoxazoline moiety and a pharmaceuticallyacceptable carrier, wherein the isoxazoline moiety forms a stablecovalent interaction with at least one amino acid residue of an MIFprotein. Preferably, the interaction occurs at or near the active siteof the tautomease activity of the MIF protein. Preferably, the compoundis an isoxazoline-containing compound or anhydroxyphenyl-isoxazoline-containing compound. More preferably, thecompound is an ester of(R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic acid,particularly the methyl ester (ISO-1). Still more preferably thecompound is and ester of2-[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-3-phenyl-propinoicacid, particularly the methyl ester (ISO-2).

As an example of the methods of treatment of the present invention,isoxazoline-containing compounds of the present invention can be used totreat patients with ARDS (acute respiratory distress syndrome). ARDS isoften considered to be an archetypal clinical response in which thedynamic balance within the immune response shifts toward excessiveinflammation and tissue destruction. MIF is expressed in both type IIalveolar cells and infiltrating immune cells. MIF levels in thebronchoalveolar lavage of ARDS patients were found to be significantlyelevated when compared to control subjects (Donnelly, et al., Nat. Med.,3, 320-323 (1997)). Human MIF enhances both TNFα and IL-8 secretion fromARDS alveolar macrophages (ex vivo) when compared to control cells.Pre-treatment of these cells with anti-MIF antibodies significantlydecreases TNFα and IL-8 production from ARDS alveolar cells. Moreover,as discussed above under “Background of the Invention,” rMIF(recombinant MIF) was found to override, in a concentration-dependentfashion, glucocorticoid-mediated inhibition of cytokine secretion inARDS macrophages. These were the first data to indicate that theMIF/glucocorticoid dyad is active in cells that had undergonepro-inflammatory activation in vivo during human disease (Donnelly, etal., Nat. Med., 3, 320-323 (1997). Significantly elevated levels ofalveolar MIF were found in those at-risk patients who progressed to ARDScompared to those who did not. MIF likely acts as an important mediatorto promote and sustain the pulmonary inflammatory response in ARDS. Itsprominent expression in ARDS may explain the fulminant course of thisdisease and perhaps why glucocorticoid treatment has provendisappointing in established cases. Thus, pharmaceutical compositionscomprising isoxazoline-containing compounds of the present invention canbe used to treat ARDS patients.

As a further example of the methods of treatment of the presentinvention, isoxazoline-containing compounds of the present invention canbe used to treat patients with rheumatoid arthritis. Synovial fluidobtained from the affected joints of patients with rheumatoid arthritiscontain significantly greater levels of MIF than those obtained frompatients with osteoarthritis or from normal control subjects (Metz, etal., Adv. Immunol., 66, 197-223 (1997); Leech, et al., Arthritis Rheum.,41, 910-917 (1998); Onodera, et al., Cytokine, 11, 163-167 (1999)). Asrevealed by immunohistochemical staining methods, infiltratingmononuclear cells within the human arthritic joint are the primarysource of MIF. In two animal models of arthritis, neutralizing anti-MIFmAb's significantly inhibited disease progression and disease severity(Leech, et al., Arthritis Rheum., 41, 910-917 (1998); Mikulowska, etal., J. Immunol., 158, 5514-5517 (1997)) giving impetus to thedesirability of developing additional MIF inhibitors for potentialtherapeutic use in inflammatory disease. Thus, pharmaceuticalcompositions comprising isoxazoline-containing compounds of the presentinvention can be used to treat arthritis patients.

In yet a further example of the methods of treatment of the presentinvention, isoxazoline-containing compounds of the present invention canbe used to treat patients with atopic dermatitis. Atopic dermatitis is achronic pruritic inflammatory skin disorder. Its pathogenesis, in part,is thought to be due to dysregulated cytokine production by peripheralmononuclear cells. In lesions from patients with atopic dermatitis, MIFprotein is diffusely distributed throughout the entire epidermal layerwith increased expression by keratinocytes (Shimizu, et al., FEBS Lett.,381, 199-202 (1996)). In normal human skin, MIF has primarily beenlocalized to epidermal keratinocytes. The serum MIF level of atopicdermatitis patients were 6-fold higher than in control subjects.Additionally, serum MIF levels in atopic dermatitis patients decreasedas clinical features improved, suggesting that MIF plays a pivotal rolein the inflammatory response in the skin during dermatitis. Thus,pharmaceutical compositions comprising isoxazoline-containing compoundsof the present invention can be used to treat patients with atopicdermatitis.

In a similar manner, the present invention also provides a method fortreating or preventing other inflammatory or autoimmune disordersincluding, but not limited to, proliferative vascular disease,cytokine-mediated toxicity, sepsis, septic shock, psoriasis,interleukin-2 toxicity, asthma, MIF-mediated conditions,insulin-dependent diabetes, multiple sclerosis, graft versus hostdisease, lupus syndromes, and other conditions characterized by local orsystemic MIF release or synthesis.

In yet another example of the methods of treatment of the presentinvention, isoxazoline-containing compounds of the present invention canbe used to treat patients with tumor growth. Neutralizing anti-MIFantibodies have been found to significantly reduce growth andvascularization (angiogenesis) of mouse 38C13 B cell lymphoma in vivo(Chesney, et al., Mol. Med., 5, 181-191 (1999)). MIF was expressedpredominantly in tumor-associated neovasculature. Cultured microvascularendothelial cells, but not 38C13 B cells, were observed both to produceMIF and to require its activity for proliferation in vitro (Takahashi,et al., Mol. Med., 4, 707-714 (1998)). In addition, the administrationof anti-MIF antibodies to mice was found to significantly inhibit theneovascularization response elicited by Matrigel implantation, a modelof new blood vessel formation in vivo (Bozza, et al., J. Exp. Med., 189,341-346 (1999)). These data indicate that MIF plays an important role intumor angiogenesis, a new target for the development of anti-neoplasticagents that inhibit tumor neovascularization.

Thus, the present invention also provides a method for treating orpreventing tumor growth or angiogenesis, comprising administering aneffective amount of a compound, or combination of compounds, having anisoxazoline moiety and that forms a stable interaction with at least oneamino acid residue of an MIF protein. Preferably, the interaction occursat or near the active site of the tautomease activity of the MIFprotein. Preferably, the compound is an isoxazoline-containing compoundor an hydroxyphenyl-isoxazoline-containing compound. More preferably,the compound is an ester of(R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic acid,particularly the methyl ester (ISO-1). Still more preferably thecompound is and ester of2-[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-3-phenyl-propinoicacid, particularly the methyl ester (ISO-2).

The present invention also provides a compound of Formula I, or apharmaceutically acceptable salt thereof, as a pharmaceuticalcomposition comprising either of the aforesaid, for use in a medicine orfor the manufacture of a medicament for the treatment or prevention ofinflammatory disorders including arthritis, proliferative vasculardisease, ARDS, cytokine-mediated toxicity, sepsis, septic shock,psoriasis, interleukin-2 toxicity, asthma, MIF-mediated conditions,autoimmune disorders (including, but not limited to, rheumatoidarthritis, insulin-dependent diabetes, multiple sclerosis, graft versushost disease, lupus syndromes), tumor growth or angiogenesis, or anycondition characterized by local or systemic MIF release or synthesis.

Pharmaceutical Formulations

The compounds of the present invention have utility in pharmacologicalcompositions for the treatment and prevention of many diseases anddisorders characterized by an MIF response, whereby MIF is released fromcellular sources and MIF production is enhanced. A compound of theinvention can be administered to a human patient by itself or inpharmaceutical compositions where it is mixed with suitable carriers orexcipients at doses to treat or ameliorate various conditionscharacterized by MIF release. A therapeutically effective dose furtherrefers to that amount of the compound sufficient to inhibit MIFtautomerase activity and MIF bioactivity, it being understood that suchinhibition may occur at different concentrations such that a personskilled in the art could determine the required dosage of compound toinhibit the target MIF activity. Therapeutically effective doses may beadministered alone or as adjunctive therapy in combination with othertreatments, such as steroidal or non-steroidal anti-inflammatory agents,or anti-tumor agents. Techniques for the formulation and administrationof the compounds of the instant application may be found in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latestaddition.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, and optionallyin a depot or sustained release formulation.

Furthermore, one may administer a compound of the present invention in atargeted drug delivery system, for example in a liposome.

The pharmaceutical compositions and compounds of the present inventionmay be manufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, dragee-making, levitating, emulsifying,encapsulating, entrapping, or lyophilizing processes. Pharmaceuticalcompositions for use in accordance with the present invention thus maybe formulated in conventional manner using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries thatfacilitate processing of the active compounds into preparations, whichcan be used pharmaceutically. Proper formulation is dependent upon theroute of administration chosen.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers,such as Hank's solution, Ringer's solution, or physiological salinebuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants areknown in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known to those in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by combining the compound with a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Liposomes and emulsions are well known examples of delivery vehicles orcarriers for hydrophobic drugs. Certain organic solvents such asdimethylsulfoxide also may be employed, although usually at the cost ofgreater toxicity. Additionally, the compounds may be delivered using asustained-release system, such as semipermeable matrices of solidhydrophobic polymers containing the therapeutic agent. Various forms ofsustained-release materials have been established and are well known bythose skilled in the art. Sustained-release capsules may, depending ontheir chemical nature, release the compounds for a few weeks up to over100 days. Depending on the chemical nature and the biological stabilityof the therapeutic reagent, additional strategies for proteinstabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid- orgel-phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

Many of the compounds of the invention identified as inhibitors of MIFactivity may be provided as salts with pharmaceutically compatiblecounterions. Pharmaceutically compatible salts may be formed with manyacids, including but not limited to hydrochloric, sulfuric, acetic,lactic, tartaric, malic, succinic, etc.; or bases. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. Examples of pharmaceutically acceptable salts, carriersor excipients are well known to those skilled in the art and can befound, for example, in Remington's Pharmaceutical Sciences, 18thEdition, A. R. Gennaro, Ed., Mack Publishing Co., Easton, Pa. (1990).Such salts include, but are not limited to, sodium, potassium, lithium,calcium, magnesium, iron, zinc, hydrochloride, hydrobromide,hydroiodide, acetate, citrate, tartrate and malate salts, and the like.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve their intended purpose. More specifically, atherapeutically effective amount means an amount effective to prevent orinhibit development or progression of a disease characterized by MIFrelease and production in the subject being treated. Determination ofthe effective amounts is well within the capability of those skilled inthe art, especially in light of the detailed disclosure provided herein.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially fromtautomerase inhibition assays and cell culture assays. Such informationcan be used to more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in a reduction in the development or severity of a diseasecharacterized by MIF release and production. Toxicity and therapeuticefficacy of such compounds can be determined by standard pharmaceutical,pharmacological, and toxicological procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds that exhibit high therapeutic indices(ED₅₀>LD₅₀ or ED₅₀>>LD₅₀) are preferred. The data obtained from cellculture assays or animal studies can be used in formulating a range ofdosage for use in humans. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (Seee.g. Fingl, et al. (1975), in The Pharmacological Basis of Therapeutics,Chapter. 1 page 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain thedesired modulating effects, or minimal effective concentration (MEC).The MEC will vary for each compound but can be estimated from in vitrodata; e.g., the concentration necessary to achieve a 50-90% inhibitionof MIF activity. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. However, HPLCassays, bioassays or immunoassays can be used to determine plasmaconcentrations.

Dosage intervals can also be determined using the MEC value. Compoundsshould be administered using a regimen that maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

In cases of local administration for instance, direct introduction intoa target organ or tissue, or selective uptake, the effective localconcentration of the drug may not be related to plasma concentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, on the subject'sage, on the severity of the affliction, on the manner of administration,and on the judgment of the prescribing physician.

The compositions may, if desired, be presented in a pack or dispenserdevice that may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. Compositions comprisinga compound of the invention formulated in a compatible pharmaceuticalcarrier may also be prepared, placed in an appropriate container, andlabeled for treatment of an indicated condition.

Materials and Methods

Synthesis. In the examples of the syntheses that follow, all reagentsand solvents used were purchased at the highest commercial quality. Allsolvents used were HPLC grade from Fisher. ¹H (270 MHz) and ¹³CNMR (67.5MHz) NMR spectra were recorded on a JEOL Eclipse 270 spectrometer.Coupling constants were reported in Hertz (Hz), and chemical shifts werereported in parts per million (ppm) relative to tetramethylsilane (TMS,0.0 ppm) with CDCl₃, DMSO or CD₃OD as solvent. Thin layer (TLC) andflash column chromatography were performed using Alumina B, F-254 TLCplates from Selecto Scientific and Fisher Scientific Basic aluminaBrockman activity I, respectively. The reactions were monitored by TLCand ¹HNMR and were stopped when the yield of the crude according to¹HNMR was 90-95%.

Reagents. Unless otherwise indicated, all chemicals were purchased fromAldrich or Sigma Chemical Companies, and were of the highest gradecommercially available. p-hydroxyphenyl-isoxazoline methyl ester wassynthesized in three steps as described previously (Xue, et al., J. Med.Chem., 40, 2064-2084 (1997); Wityak et al., J. Med. Chem., 40, 50-60(1997); Baraldi, et al., Synthesis—Stuttgart—1994, 11, 1158-1162 (1994);Curuzu et al., Collect. Czech. Chem. Commun., 56, 2494-2499 (1991),which can be summarized as follows (FIG. 1): 4-hydroxy benzaldehyde (4.0g, 32.8 mmol) and hydroxylamine hydrochloride (2.28 g, 32.8 mmol) weredissolved in methanol (100 mL) followed by addition of sodium carbonate(6.95 g, 65.6 mmol). Overnight reaction gave product 2 in 95% yield (4.3g). Chlorination of the oxime 2 using N-chlorosuccinimide (4.22 g, 31.6mmol) in DMF (100 mL) quantitatively furnished chlorooxime 3. Compound 3was then dissolved in THF/water (80/20) and treated with 3-butenoatemethyl ester (3 g, 24.5 mmol) and sodium carbonate (7.8 g, 73.6 mmol).After completion (12 hr), the product was extracted with ethyl acetateand the organic extracts were washed with brine and dried over magnesiumsulfate. Flash chromatography afforded the product, 4, in 75% yield(0.42 g). The structure was confirmed by ¹H— and ¹³C-NMR and massspectroscopy.

Dopachrome methyl ester was prepared similarly to previously publishedprocedures (Bendrat, et al., Biochemistry, 36, 15356-15362 (1997);Swope, et al., EMBO J., 17, 3534-3541 (1998)). Briefly, to an aqueous 4mM solution of L-3,4-dihydroxyphenylalanine methyl ester was added NaIO₄to a final concentration of 6 mM. The solution was immediately placed onice. Assays were initiated at a time when the absorbance at 475 nmreached a maximal value, signifying that the limiting reagent, NaIO₄,was consumed. Recombinant human and mouse MIF was expressed in E. coliand purified as previously reported (Bernhagan, et al., Biochemistry,33, 14144-14155 (1994).

Treatment of MIF with Inhibitors. MIF samples (procedure A: 0.72 μg/mlin 50 mM sodium phosphate (pH 6.6) containing 20 μg/ml bovine serumalbumin; procedure B: 0.72 μg/ml in 50 mM sodium phosphate (pH 6.6)containing fetal bovine serum; procedure C: 0.1-0.6 mg/ml in 50 mMsodium phosphate at pH 6.6) were treated with various concentrations ofthe inhibitors for 5-20 minutes (exact times are specified in the textof the Examples below) at room temperature. Treated MIF samples werethen analyzed for enzyme activity using the dopachrome tautomeraseassay. Protein concentrations were determined using the micro BCA assay(Pierce Chemical Co.).

Dopachrome Tautomerase Assays. To a room temperature solution (0.7 ml)of recombinant mouse or human MIF samples (0.72 μg/ml in the specifiedbuffers from procedures A, B, and C above) was added dopachrome methylester (0.3 ml at 4 mM, prepared in situ). The sample was immediatelymonitored for loss in absorbance at 475 nm compared to untreated MIFsolutions and to dopachrome methyl ester without the addition of MIF.

MALDI MS Experiments. Samples were run using a Perceptive Voyager DEMALDI MS (DHB matrix) at the University of Washington Department ofBiochemistry Mass Spectrometry Laboratory.

Example 1

Enzyme Inhibition Studies. This example illustrates the inhibition ofthe enzymatic activity of human MIF by isoxazolines. The enzymatictautomerization activity of recombinant human MIF was performed usingL-dopachrome methyl ester as a chromogenic substrate (Bendrat, et al.,Biochemistry, 36, 15356-15362 (1997)). The tautomerization reactioncatalyzed by MIF, as described in detail above, leads to the formationof a dihydroxyindole product which is colorless.

Several isoxazoline derivatives were prepared and tested for activity inthe MIF dopachrome tautomerase assay. Compound 4 (FIG. 1) inhibited MIFtautomerase activity in a dose-dependent manner with an IC₅₀ of about 10μM, but the corresponding non-hydroxylated phenyl analog was about 12times less potent. The 4-methoxy analog (7; FIG. 3) showed no activity,reinforcing an earlier conclusion that a para hydroxyl function is animportant feature of our emerging pharmacophore for MIF tautomeraseinhibitors, probably attributable to the advantageous formation of ahydrogen bond with Asp97 as suggested by the MIF:phenylpyruvateco-crystallization data.

To continue structure:activity relationship (SAR) testing in theisoxazoline series, the isoxazoline test compound 4 was oxidized toisoxazole 5 (Xue, et al., J. Med. Chem., 40, 2064-2084 (1997)), as shownin FIG. 2, which eliminated the chiral center, offering a solution tothe problem of enantiomeric preference in the isoxazoline series.Surprisingly, the isoxazole 5 was totally inactive in the dopachrometautomerization assay. As shown in X-ray studies of a crystallineco-structure of MIF and racemic mixture of compound 4, that only the Sstereoisomer binds to MIF. To purify the stereoisomers, the enantiomersare conveniently isolated by a chiral separation process using HPLC(Wityak, et al., J. Med. Chem., 40, 50-60 (1997)).

Functionalized Isoxazolines. This section describes a new strategy thataims at improving the binding of the p-hydroxyphenyl ring to the activesite of MIF. The new synthesis is begun with either mono- ormulti-substitution of the p-hydroxybenzaldehyde ring with group(s) suchas —OH, —SH, —CN, —NHAc, —N₃ and halides. Initially, structural activityrelationships are created at positions 2, 3, 5, and 6 utilizing onlycommercially available material to synthesize isoxaline derivatives.Synthesis of any starting material needed to serve this aim isconveniently done after close examination of the first set ofisoxazoline analogs by measuring the MIF tautomerase activity, asdescribed above.

In previous experience with amino acid Schiff base-type compoundsdescribed previously (see U.S. application Ser. No. 09/699,258, supra),the side chain of the amino acid residues played an important roll inimproving the binding affinity in the vicinity of the hydrophobic core,by an unidentified mechanism. For instance, in the dopachrometautomerization assay, the IC₅₀ of glycine and tryptophan Schiff basederivatives is 100 μM and 1.6 μM respectively. Initially, a logicalapproach to improve potency of compound 4 (ISO-1) is functionalizationat the alpha position to produce a wide diversity of novel compounds. Aninitial compound has been synthesized, namely,2-[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-3-phenyl-propinoicacid methyl ester (ISO-2), as a mixture of four diastereomers. Uponfractionation, one fraction, a mixture of two isomers, was found toinhibit MIF dopachrome tautomerase activity with an IC₅₀ 550 nM (FIG.4). However, this synthetic route requires preparation of functionalizedalkenes as starting materials (not available commercially) and producesa mixture of four diastereomers. This direct approach therefore requireschiral HPLC separation and, therefore, is less preferred than the aboveapproach of studying structural activity relationships at positions 2,3, 5, and 6 utilizing only commercially available material to synthesizeisoxaline derivatives.

Another approach to functional derivatives of compound 4 begins withpure enantiomer and can be summarized as shown in FIG. 5. This approachprovides diversity around the amino functional group and also produces aseparable mixture of two diastereomers. This strategy is achieved bystarting from R- or S-vinylglycine, both of which are commerciallyavailable. Functionalization of each isomer is accomplished by eitherreductive amidation using a wide range of aliphatic or aromaticaldehydes, or by amidation via coupling with aliphatic or aromatic acid.Moreover, Pd-mediating aryl halides coupling to produce aryl amines alsoare used in this approach. This approach generates diversity around theamino functions and is based on similar synthetic routes that are wellknown in the art.

Thus, according to the present invention, the isoxazoline-basedcompounds related in structure to compound 4 comprise a new and generalclass of low molecular weight, specific inhibitors of MIF enzymaticactivity.

Example 2

Biological Assay of MIF Activity. This example shows thatisoxazoline-based compounds not only specifically inhibit MIF enzymaticactivity, but also inhibit MIF immunoregulatory activities,specifically, MIF glucocorticoid regulating activity. The ability ofp-hydroxyphenylisoxazoline methyl ester to neutralize the effect of MIFto influence the anti-inflammatory effect of dexamethasone on TNFαproduction by human monocytes was tested. As shown in FIG. 3,p-hydroxyphenylisoxazoline methyl ester significantly inhibited theMIF-dependent interference with glucocorticoids in this test system.This property of isoxazoline was dose dependent with an IC₅₀ of 5 μM. Toaddress the specificity of this inhibitory effect on MIF, otherisoxazoline analogs were tested (e.g., non-hydroxylated form andcompounds 5, 6 and 7) that are not such potent inhibitors of MIFtautomerase activity and found that these compounds do not provide anyanti-inflammatory activity (IC₅₀>100 μM for all), in contrast to theisoxazoline compound 4. These results are consistent with a hypothesisthat the pro-inflammatory effects of MIF can be neutralized by thebinding of a small molecule at the tautomerase active site, althoughthis effect is not believed to depend on the neutralization oftautomerase activity per se.

The compounds are additionally assessed for inhibition of MIF biologicalactivities in any of a number of assays for MIF biological activityincluding, for example, inhibition of MIF binding to target cells,inhibition of MIF release or synthesis, inhibition of MIFimmunoreactivity with MIF-specific antibodies, alterations of MIFconformation or structural integrity as assessed by circular dichroismspectroscopy, liquid NMR-spectroscopy, X-ray crystallography, thermalstability measurement, inhibition of the pro-proliferative effects ofMIF on quiescent NIH/3T3 cells and inhibition of the associatedprolonged ERK activation therein, inhibition of MIF-induced arachadonicacid release from NIH/3T3 cells, inhibition of MIF-induced fructose 2,6bisphosphate formation in L6 myocytes, inhibition of MIF toxicity in theMIF, TNF, or LPS-challenged test animals, inhibition of theglucocorticoid counter-regulatory activity of MIF in vitro or in vivo,inhibition of the MIF-induced functional inactivation of the p53 tumorsuppressor protein (Hudson, et al., J. Exp. Med., 190, 1375-1382 (1999),inhibition of MIF-induced release of prostaglandin E2, and inhibition ofmorbidity or mortality in any of a number of animal models of humandiseases that are characterized by the release, production and/orappearance of MIF.

Example 3

Co-crystal structure analysis of MIF: isoxazoline 4 (ISO-1). Recently,the crystal structure of MIF complexed with p-hydroxyphenylisoxazoline 4has been resolved. Similarly to p-hydroxyphenylpyruvate (Lubetsky, etal., Biochemistry, 38, 7346-7354 (1999)) and(E)-2-fluoro-p-hydroxycinnamate (Taylor et al., Biochemistry, 38,7444-7452 (1999)) co-crystallization with MIF, three molecules ofisoxazoline bind each MIF trimer molecule and lie at each interfacebetween two subunits. The inhibitor interacts with Pro-1 and Lys-32 fromone subunit and Asn-97 from the adjacent unit. In particular, theinhibitor 4 interacts with Pro-1 of MIF via the C3 carbon of theisoxazoline ring.

Structural analysis of the bound molecules revealed that only theS-isomer can bind to the MIF cleft, supporting earlier indications thatthere were likely to be substantial enantiomeric effects in some racemicmixtures of possible inhibitor compounds. Moreover, reduction of thecarboxylate moiety of p-hydroxyphenylisoxazoline to furnish the alcoholadduct 6 abolishes the inhibitory effect on the tautomerase activity inan agreement with the postulated importance of this group to formhydrogen bonds with Lys-32.

1. A method for inhibiting enzymatic and biological activity of humanMIF comprising contacting the human MIF with a compound, or combinationof compounds, having an isoxazoline moiety that forms a stableinteraction with at least the N-terminal proline residue of the humanMIF.
 2. The method of claim 1, wherein said compound is anisoxazoline-containing compound of Formula I, wherein Formula I is:

wherein: R₁₋₄ are, independently, R, halo, N₃, CN, OH, NRR′, or SH; Rand R′ are, independently, H or C₁₋₆ alkyl; X is R, halo, N₃, CN, OR,NRR′, SH, ═O, ═CH₂, or A; A is a substituted or unsubstituted aromaticring; Y is R, NRR′, NRR″ or (CH₂)_(n)-A; Z is R, OR, OR″, NRR′, NRR″, orA; R″ is a saturated or unsaturated, straight or branched chain C₂-C₁₈;and n is 0 or
 1. 3. The method of claim 2, where said compound is apara-hydroxyphenyl-isoxazoline-containing compound, wherein each of R,R₁₋₄, X and Y is H or —CH₂-A, and Z is OR.
 4. The method of claim 3,wherein the compound is an ester of(R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic acid methylester (ISO-1).
 5. The method of claim 4, wherein the compound is(R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic acid methylester (ISO-1).
 6. The method of claim 3, wherein said compound is anester of2-[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-3-phenyl-propinoicacid methyl ester (ISO-2).
 7. The method of claim 6, wherein saidcompound is2-[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-3-phenyl-propinoicacid methyl ester (ISO-2).
 8. A method a method for treatinginflammatory disorders or any condition characterized by local orsystemic MIF release or synthesis, comprising administering an effectiveamount of a compound of Formula I to a subject in need thereof, whereinFormula I is:

wherein: R₁₋₄ are, independently, R, halo, N₃, CN, OH, NRR′, or SH; Rand R′ are, independently, H or C₄, alkyl; X is R, halo, N₃, CN, OR,NRR′, SH, ═O, ═CH₂, or A; A is a substituted or unsubstituted aromaticring; Y is R, NRR′, NRR″ or (CH₂)_(n)-A; Z is R, OR, OR″, NRR′, NRR″, orA; R″ is a saturated or unsaturated, straight or branched chain C₂-C₁₈;and n is 0 or
 1. 9. The method of claim 8, wherein each of R, R₁₋₄, Xand Y is H or —CH₂-A, and Z is OR.
 10. The method of claim 9, whereinsaid disorder or condition is selected from the group consisting ofatopic dermatitis, arthritis, proliferative vascular disease, ARDS(acute respiratory distress syndrome), cytokine-mediated toxicity,sepsis, septic shock, psoriasis, interleukin-2 toxicity, asthma,MIF-mediated conditions, autoimmune disorders, tumor growth orangiogenesis.
 11. The method of claim 10, wherein said autoimmunedisorder is selected from the group consisting of rheumatoid arthritis,insulin-dependent diabetes, multiple sclerosis, graft versus hostdisease, and lupus syndromes.
 12. A pharmaceutical compositioncomprising a compound of Formula I, or a pharmaceutically acceptablesalt thereof, and a pharmaceutically acceptable carrier or diluant,wherein Formula I is:

wherein: R₁₋₄ are, independently, R, halo, N₃, CN, OH, NRR′, or SH; Rand R′ are, independently, H or C₁₋₆ alkyl; X is R, halo, N₃, CN, OR,NRR′, SH, ═O, ═CH₂, or A; A is a substituted or unsubstituted aromaticring; Y is R, NRR′, NRR″ or (CH₂)_(n)-A; Z is R, OR, OR″, NRR′, NRR″, orA; R″ is a saturated or unsaturated, straight or branched chain C₂-C₁₈;and n is 0 or
 1. with the provisos that: (a) when R₁=R₂=tert-butyl andeach is ortho to OR, at least one of R, R₃, R₄, X and Y is not H or Z isnot OCH₃; and (b) when Z=OH and R=H or methyl, at least one of R₁₄, Xand Y is not H.
 13. The pharmaceutical composition of claim 12, whereinsaid compound is an ester of(R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic acid.
 14. Thepharmaceutical composition of claim 13, wherein said compound is(R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolineacetic acid methylester (ISO-1).
 15. The pharmaceutical composition of claim 12, whereinsaid compound is an ester of2-[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-3-phenyl-propinoicacid.
 16. The pharmaceutical composition of claim 15, wherein saidcompound is2-[3-(4-hydroxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-3-phenyl-propinoicacid methyl ester (ISO-2).